1.
Presentation on Production of
Enzyme -
Lipase

2.
Contents
Introduction
Historical Background
Properties
Reactions Catalyzed
Classification
Sources
Production Technology
Submerged Fermentation
Solid-State Fermentation
Downstream Processing
Factors affecting production
Applications

3.
INTRODUCTION
• Lipases are hydrolases capable of catalyzing the hydrolysis of
Triglycerols (TAGs) into Glycerol and Fatty acids (FAs).
• These enzymes operate at the interfaces of Biphasic systems, which
is a phenomenon known as interfacial activation.
• These do not require co-factors and are easily immobilized on
different matrices.
• The active sites of lipases are generally characterized by amino acid
triad composed of serine, histidine and aspartate.
• Lipases exihibit region-selective properties and enantioselective
catalytic behaviour and are considered to be the most versatile
catalyst in lipid biotechnology.
• These enzymes can be employed in a large number industrial
processes ( production of agrochemicals, cosmetics , biodiesel etc.)

4.
HISTORICAL BACKGROUND
• In 1856, Claude Bernard first discovered a lipase in pancreatic juice as an enzyme
that hydrolyzed insoluble oil droplets and converted them to soluble products.
• In 1901, the presence of lipases has been observed for Bacillus prodigiosus ,
B.pycocyancus and B.fluorescens which represents today’s best studied lipase
producing bacteria now named Serratia marcescens , Pseudomonas aeruginosa
and P.fluorescens.
• Lipase have traditionally been obtained from animal pancreas and are used as a
digestive aid for human consumption either in crude mixture with other hydrolases
(pancreatin) or as a purified grade.
• Lipolase was the first commercial recombinant lipase industialized from the
fungus Thermomycesl anugiwnosus and expressed in Aspergillus oryzae in 1994.

5.
PROPERTIES
• pH optima
• Temperature optima and thermal inactivation
• Activation and inactivation of the enzyme
• Substrate specificity

6.
Reactions catalyzed

7.
Classification

8.
SOURCES
Plant lipases:
These have been isolated from the
leaves, oils, latex and seeds of
oleaginous plants and cereals.
Yeast Lipases:
These include species Candida
antartica, Candida rugosa, Candida
utilis and Saccharomyces species. The
production of Biodiesel includes lipases
from Thermomycesl anuginosus.
Image Source: Research Gate Image Source: Wiley Online Library

9.
Fungal Lipases:
Filamentous fungi are
considered to be the best
source for production of
lipases. The genera
includes Aspergillus,
Rhizopus , Penicillium ,
Mucor, Geotrichum and
Yarrowia etc.
Bacterial Lipases:
The genera Pseudomonas
and Burkholderia are the
most widely used for the
production of bacterial
lipases. P.aeruginosa
produces a cystiene
hydrolase solvent tolerant
lipase.
Animal Lipases:
These include pancreatic
and pregastric lipases.
Porcine and Human
pancreas were the first
sources of lipases used
in food processing.
Image Source: Health Jade Source: Microbewiki -Kenyon College Source: Research Gate

10.
PRODUCTION TECHNOLOGY
• Screening
• Strain selection
• Inoculum
preparation
• Immobilization
• Solid-State
Fermentation
• Submerged
Fermentation
• Filtration
• Centrifugation
• Chromatography
• Aqueous two
phase
Upstream
Processing
Production
Downstream
Processing

11.
Raw Materials and Nutrients
• Olive oil, Palm oil,
Coconut oil
• wheat Bran, rice bran
• yeast extract, peptone
• Urea, NaNO2
• Sucrose , glucose ,
fructose
• KH2PO4
• MgSO4 .7 H2O
Microbial Sources
Aspergillus sp.
Bacillus sp
Candida antarctica
Pseudomonas aeruginosa
Staphylococcus aureus
Aspergillus awamori
Bacillus subtilis
Aureobasidium pullulans
Yarrowia lipolytica

12.
PRODUCTION
• Production can be carried out through two modes:
• Submerged Fermentation
• Solid-State Fermentation
• Submerged fermentation could be conducted by batch, but productivity is
increased by the fed batch or continuous processes.
• Greater enzyme production is obtained using defatted soyabean flour as a
substrate.
Submerged Fermentation

13.
Solid-State Fermenation
• Lipases produced had greater thermal stability.
• Use of agro-industrial residues as substrates greatly reduces the cost of production.

14.
Conditions For SmF
• Species: Aspergillus sp.
• Carbon Sources: Fructose, Glucose , galactose
• Inducers: Olive oil , palm oil , corn oil , coconut oil
• Nitrogen Sources: Yeast extract , peptone , urea ,
tryptone , NaNO2
• Surfactants: Tween-80 , SDS , TritonX-100 , C-TAB
• Substrates: Wheat bran , rice bran , wheat straw,
pectin , xylan
• pH: 6.0-7.2
• optimum temperature: 37o C
• Incubation Time: 24-48 hours
• Corn steep liquor,K2HPO4,KH2PO4
• MgSO4.7H2O.
Image Source : Research Gate

15.
Conditions For SSF
• Species: Aspergillus sp.
• Carbon sources: glucose, fructose, galactose
• Inducers: olive oil , soyabean oil , cococnut oil
• Nitrogen sources: Urea , yeast extract ,
ammonium sulphate , sodium nitrate
• Support: sugarcane bagasse
• Substrates: lignocellulose, wheat straw, rice
straw, soyabean bran, lemon peel
• pH: 6
• Optimum temperature: 35- 40 oC
• Incubation time : 72 hours
• Rice husk, KH2PO4 , MgSO4
Image Source : Research Gate

16.
Maximum Growth Period
• The lipase production occurs during
the late logarithmic phase or
stationary phase.
• Thus, the cultivation period may vary
according to the microorganism and its
growth rate.
• Inducers also induce and increased
production of lipases, such as free
fatty acids, hydrolyzable esters, bile
salts and glycerol.

17.
Fermentation by Cell Immobilization
• Enzyme stability and activity of lipases can be improved through the
immobilization process.
• The immobilization process is advantageous, as it can prevent biomass washout at
high dilution rates.
• The separation of biomass from the medium is favored due to the high cell
concentration in the reactor.
• For example: Cell growth and immobilization of Rhizopus oryzae fungus cells for
the production of biodiesel through methanolysis of soybean oil.
• Fibrous nonwoven fabric used as the immobilization matrix in a circulating
packed-bed bioreactor.

18.
Downstream Processing
• Concentration
• Chromatography
• Aqueous Two-Phase Systems
• NOVEL PURIFICATION STRATEGIES
• Reverse Micellar System
• Membrane Processes
• Extractive Fermentation

19.
• Reverse micellar extraction is a
liquid–liquid extraction method that
uses an organic solvent containing
water droplets stabilized by a layer of
surfactant molecules (CTAB).
Membrane Processes
• The polyvinylidene fluoride,regenerated
cellulose and glass fiber membranes are
usually applied in microfiltration and
ultrafiltration.
• The principle of membrane separation is
based on the different permeability of
substances through the membrane material
and the driving force for the separation is
either pressure, or a difference in
concentration, and/or electric potential.
Reverse Micellar Systems
Source
:
Research
Gate
Source
:
Science
Direct.com

20.
Aqueous Two-Phase Systems
• The two immiscible aqueous phases of an aqueous two phase
system (ATPS) are considered an ideal liquid–liquid
purification technique for the separation, concentration and
extraction of biomolecules due to the high productivity,
simplicity, short processing time, scalability, cost
effectiveness and versatility of the system.
• An ATPS consists of a mixture of polymers and salts.
• The polymers can be dextran, polyethylene glycol and
polypropylene glycol. The salts can be phosphates, sulfates,
surfactants (n-decyl tetraethylene oxide and octylphenol
ethoxylate) and ionic liquids (1-ethyl-3-methylimidazolium
acetate and 1-butyl-3-methylimidazolium
hexafluorophosphate). Low-molecular-weight alcohols (i.e.,
ethanol and propanol) may also be present.
Image Source : Science direct.com

21.
Improved Lipases-Genetic Recombination
• Modern genetic recombination systems are used for insertion of gene of interest
into the microorganisms to enhance the production.
• For Example:
• A bacterial lipase gene from Bacillus subtilis was expressed in Saccharomyces
cerevisiae and a significant increase in lipase production was observed.
• A lipase gene (Lip) of the Aspergillus niger was de novo synthesized and
expressed in the Trichoderma reesei under the promoter of the cellobiohydrolase I
gene (cbh1).
• Expression of cellobiohydrolase was suppressed via RNAi method.
• The reconstructed strains with decreased CBHI production exhibited increase in
lipase production than that of parental strain.

22.
Methods for detection of microbial lipase
production
• Different techniques have been developed for the screening of microorganisms for
lipase production.
• Qualitative screening of microorganisms on selective growth media:
• In this technique, lipolysis is detected by changes in the appearance of the
substrates (such as tributyrin and triolein) that are emulsified in the growth media.
• The formation of clear halos around the colonies cultivated on the agar plate is an
indication of lipase production.
• For Example : Lipolytic Bacillus sp. LBN 4 was isolated on tributyrin agar medium
using glycerol tributyrate as substrate.
• Solid media supplemented with dyes such as phenol red, Victoria Blue B, Spirit
blue, or Nile blue sulfate as pH indicators are also used for determination of
lipolytic activity. For example : used for screening of Bacillus strain.

23.
Quantitative titrimetric assay
• Lipase activity is measured quantitatively on a continuously stirred
triacylglyceride emulsion by neutralization of free fatty acids released following
addition of titrated NaOH (in order to maintain the pH at a constant end point
value).
• olive oil is used as a substrate for the titrimetric analysis.
• For Example :
• Lipolytic activity of Pseudomonas monteilli 2403- KY120354 was measured in a
reaction mixture containing olive oil emulsion incubated at 37 °C for 1 h. Enzyme
activity was terminated after addition of 20 mL acetone: ethanol mixture (1:1).
The liberated free fatty acids were titrated against 0.1 M NaOH using
phenolphthalein.

24.
Factors Influencing Production
Factors
Temperature
Surfactants
Agitation
Carbon
substrate
source
Nitrogen
source

25.
Applications
• Food Industry: Used in the
production of dairy products, baked
foods and fruit juices as well as the
interesterification of fats and oils to
produce modified acylglycerols.
Application Process details Species
Food Industry Synthesis of methyl acetate Candida rugosa
Food Industry Glycerolysis of corn oil Candida antarctica
A human milk fat substitute, was the
first commercial product made by
1,3-specific lipases treatment of
tripalmitin with unsaturated FAs.

26.
Biodiesel
Lipases are able to synthesize
biodiesel in the presence of high
water content, which is a useful
strategy when waste oils are used,
since they usually contain a high
amount of water molecules.
Application Process Details Species
Biodiesel Methanolysis of soybean oil for
biodiesel production
Burkholderia
ubonensis SL-4
Biodiesel Transesterification of Jatropha oil Pseudomonas
aeruginosa AAU2
Ecodiesel 100 is produced from the
partial 1,3-regiospecific alcoholysis
using pig pancreatic lipase.

27.
Bioremediation
These enzymes are able to enhance
the bioremediation of greasy
effluents containing fats, oils and
proteins discharged by the dairy
industry.
Application Process Details Species
Bioremediation Potential application in the
treatment of effluents laden
with oil (degrading ayurvedic
oil)
Aspergillus awamori
BTMFW032
The remediation of cooking oil wastes
using orange lipase for transesterification
reaction and proved less toxicity of treated
waste oils compared to untreated oils.

28.
Pharmaceutical Industry
• Use of lipases in the preparation of
optically active compounds, such as pure
alcohols, amines and carboxylic acids.
• Lipases can synthetize a chiral
intermediate compound for the production
of Polixatel (taxol 1), which has been
applied as an anticancer drug, especially
ovarian cancer.
Application Process details Species
Pharmaceutical
industry
Enantioselective esterification
toward (R)-1-(4-methoxyphenyl)-
ethanol (MOPE)
Pseudomonas stutzeri
Pharmaceutical
industry
Lipase-catalyzed degradation of
polyhydroxyalkanoate (PHA)
Bacillus subtilis

29.
Detergent industry
• Lipolase from Thermomyces lanuginosus
represents the first industrial lipase to be
introduced into detergent and was
commercialized in 1988 by Novo Nordisk.
• Other lipases including Lumafast
(Pseudomonas mendocina) and Lipomax
(Pseudomonas alcaligenes) were
commercialized by Genencor.

30.
REFERENCES
• Adetunji AI, Olaniran AO. Production strategies and biotechnological
relevance of microbial lipases: a review. Brazilian Journal of Microbiology.
2021 Sep;52(3):1257-69.
• Melani NB, Tambourgi EB, Silveira E. Lipases: from production to
applications. Separation & Purification Reviews. 2020 Apr 2;49(2):143-58.
• Geoffry K, Achur RN. Screening and production of lipase from fungal
organisms. Biocatalysis and agricultural biotechnology. 2018 Apr 1;14:241-
53.
• Sarmah N, Revathi D, Sheelu G, Yamuna Rani K, Sridhar S, Mehtab V,
Sumana C. Recent advances on sources and industrial applications of
lipases. Biotechnology progress. 2018 Jan;34(1):5-28.